Protease is a collective term for enzymes catalyzing hydrolysis of peptide bonds and is present ubiquitously in microorganisms, animals and plants. A protease is also a typical industrial enzyme and is used extensively in the fields of detergents, leather processing, food processing and functional peptide production. Particularly, from the viewpoint of prevention of secondary infection via medical apparatus, there are few alternative solutions to protease-mediated degradation of infectious protein contaminants on medical apparatus. For practical use, the most important properties of an industrial enzyme are high stability and high activity under the conditions of use. Particularly, high physicochemical stability to heat is often required and thus heat-resistant proteases are extensively used as an industrial protease. Currently, as an industrial protease, subtilisin family proteases such as subtilisin carlsberg and Proteinase K are known. In particular, Prionzyme available from the U.S. company Genencor has the highest stability among commercial proteases and is used for apparatus cleansing which is intended to prevent transmission of abnormal prion proteins, which are causative agents of prion diseases such as CJD (Creutzfeldt-Jakob disease). However, the optimum conditions for Prionzyme are limited to a temperature of 40 to 60° C. and a pH of 8 to 10, and there has been a desire for practical application of proteases usable at higher temperature conditions. Accordingly, many attempts have been made to find a novel protease usable under high temperature and high alkaline conditions.
For example, the present inventors found a subtilisin family protease derived from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 (hereinafter referred to as “Tk-subtilisin”), and reported that Tk-subtilisin shows the highest activity at pH 9.5 at a temperature of 80 to 100° C. among various conditions and has the highest thermostability among known proteases (see nonpatent literature 1 and 2). The patent literature 1 discloses a heat-resistant protease derived from the same Thermococcus kodakaraensis KOD1 and reports that the optimum temperature for this heat-resistant protease is about 80° C., that the protease has such a heat resistance that the residual activities are about 75% and about 50% after 120 minute-incubation at 70° C. and 80° C., respectively, and that the protease has such an alkali resistance that the residual activities are 90% or more and about 85% after 120 minute-incubation at pH 11 and pH 11.5, respectively.